This proposal is to study regulation and function of MAP kinase-activated protein kin ses (MAPKAPKs), which are important, expanding, and understudied components in the MAP kinase cascades. One key role of MAPKs is to control gene expression. MAPKs and MAPKAPKs collaborate in transcription, translation, and stabilization. Understanding this collaboration is vital to design of therapies in many diseases because MAP (mitogen-activated protein) kinase (MAPK) cascades are activated by extracellular signals (growth factors, hormones, cytokines) and intracellular signals (cellular stresses and checkpoints) as part of interconnected cascades of enzyme activations and inhibitions controlling metabolism, gene expression, and growth and development. Abnormal functions of the pathways is involved in cancer, proliferative complications of diabetes, and genetic disorders. There are three principal MAPKs (ERK, JNK, and p38 MAPK) but there are many isoforms. MAP kinases select specific targets for regulation, including members of a diverse group called collectively MAPKAPKs which extend the cascade. MAPKAPKs are related to each other by their C-terminal domains independent of whether they have one kinase domain (MNKs, MAPKAPK2) or two domains (RSKs, MSKs). Aim one is determine how MAP kinases bind and regulate their specific MAPKAP kinase targets. Aim two is to study the cellular functions of MAPKAPKs by surveying the mammalian proteome for phosphorylations inducible by MAPKAPKs. Aim three is to design and use recombinant activated COOH-terminal domains (CTDS) of RSKs and MSKs to characterize their CTD kinase activity independently of the NTD domain, and to characterize nuclear RSKs and MSKs by extraction and chromatography. Aim four is to characterize the activation, enzymatic properties, and functions of MAPKAP kinase-like enzymes (Rcklp, Rck2p) in yeast, a model eukaryote, using combined biochemical and genetic approaches.